Methods and compositions for improved fertilization and embryonic survival

ABSTRACT

Single nucleotide polymorphic sites of the bovine HSP genes are associated with improved fertilization rate and/or improved embryo survival rate in cattle. Nucleic acid molecules, kits, methods of genotyping and marker assisted bovine breeding methods based on these SNPs are disclosed.

STATEMENT OF GOVERNMENT INTEREST

This invention was made with government support under 09-CRHF-0-6055awarded by the USDA/NIFA. The government has certain rights in theinvention.

FIELD OF THE INVENTION

The present invention relates to a method of genetic testing forimproved fertilization rate and embryonic survival rate in animals,especially dairy cattle, based on single nucleotide polymorphisms (SNPs)in genes encoding the heat shock proteins (HSP).

BACKGROUND OF THE INVENTION

Poor reproductive performance, in particular in high-producing dairycows, is a major problem on dairy farms throughout the world and hasbeen identified as the single most important problem in dairy herdmanagement in many countries (Royal et al., 2000; Dobson et al., 2008).In addition to direct financial losses, infertility can result inincreased management complexity, for example, an inability to achieve acompact calving pattern, which is of critical importance in maximizingmilk production from grazed grass in seasonal production systems.

This is of particular interest in cattle because of declining fertilityover the past few decades (Dobson et al., 2007; Leroy et al., 2008).Furthermore, recent studies have shown that low fertilization rates andembryonic loss seem to be the main factors in dairy cattle infertility(Santos et al., 2004; Morris and Diskin, 2008).

While reproductive performance is influenced by a large number offactors, low fertilization rate and early embryonic loss are the primaryfactors contributing to poor reproductive performance in dairy cattle(Santos et al., 2004; Morris and Diskin, 2008). Enormous efforts, suchas animal breeding and artificial insemination, have been and continueto be invested in ensuring adequate fertility in the cattle herd.Typically, artificial insemination in dairy cattle is successful only30-35% of the time. The reasons for this are not clear. However, it isunderstood that both biological and environmental factors affectfertility rate. Some environmental factors such as high temperature, andlack of precipitation can cause stress in cattle and can drop thefertility rate to 10-15%. Commercial artificial insemination operationsoften shut down in July and August due to the drop in fertility causedby the hot, dry weather.

Genetics is also a prominent factor in fertility, and accounts for aboutone-third of the decline in pregnancy rate of dairy cows (Shook, 2006).In particular, identifying highly fertile bulls has been atime-consuming and expensive process. It can take 5-10 years of trackingthe attempts of artificial insemination using semen from a bull beforeit can be certified as a quality bull.

Marker-assisted selection, on the other hand, can lower the high costand reduce the extended time commitment of progeny testing currentlyused to improve sires, since young bull progeny could be evaluatedimmediately after birth or even prior to birth for the presence/absenceof the marker, and young bulls that are determined by genetic testing tohave undesirable markers would never be progeny tested.

There is thus a need for a method of genetically evaluating the bulls,as well as the cows, e.g., by genetic testing, to enable a quick andaccurate evaluation of its fertility as well as the survival rate ofembryos conceived therefrom.

Heat shock proteins (HSPs) are among the most highly conserved proteinsin nature and have been found in all organisms studied from bacteria tohumans (Becker and Craig, 1994). The structure and roles of HSPs asmolecular chaperones in the folding, transport, and assembly of proteinsas well as in protecting the cell under different stress conditions havebeen extensively studied and reported in the scientific literature(Becker and Craig, 1994; Nollen and Morimoto, 2002; Qiu et al., 2006).The fact that HSPs are essential in the folding, stability, and cellularlocalization of newly synthesized proteins implies key roles of theseproteins in apoptosis, cell differentiation, and regulation of theembryo cell cycle (Luft and Dix, 1999; Lanneau et al., 2007). Strongevidence which has accumulated on the expression of HSPs duringspermatogenesis, oogenesis, and embryogenesis suggests they haveimportant functions in fertilization and during the pre-implantationperiod (Neuer et al., 1999). Mouse embryos cultured with monoclonalantibodies to HSPs have been found to display a significantly reducedblastocyst rate (Neuer et al., 1998). Al-Katanani and Hansen (2002)reported that the addition of antibodies for the induced form of HSP70reduced blastocyst rate of cattle embryos, suggesting that HSP70 isinvolved in proper embryonic development. Matwee and colleagues (2001)showed that fertilization—measured as the number of permatozoa tightlybound to a zona pellucida—and embryo development in cattle weresignificantly affected by the presence of different concentrations ofanti-HSP70.

Although there is strong evidence in the literature on the roles of HSPsin early embryonic development, most of the studies have focused on themouse and only a few HSP genes have been studied in cattle embryos. Noevidence existed that any polymorphism in HSPs, if existed, is relatedto dairy cattle fertility in any way.

SUMMARY OF THE INVENTION

The present inventor investigated the expression profiles of HSPs andtheir splice variants in bovine embryos (degenerates vs. blastocysts),and also analyzed the association of these profiles with fertilitytraits. Some splice variants showed differential expression betweendegenerates and blastocysts while others were not expressed at all inembryos, implying different functions of these transcripts in embryonicdevelopment. Among the HSPs investigated, DNAJC15, DNAJC19, DNAJC24 andDNAJC27, all of the HSP40 family, had the most significant expressiondifferential. They were further investigated for association withfertility and development traits. Single nucleotide polymorphisms (SNP)in DNAJC15 and DNAJC27 were found to be associated with blastocyst rateand fertilization rate, respectively.

Accordingly, the present invention provides an isolated nucleic acidmolecule comprising at least one polymorphic site selected from thegroup consisting of positions 85146, 85161, 85216, 85292, and 85300 ofthe nucleic acid sequence shown in FIG. 1 (SEQ ID NO: 1) (part of theDNAJC15 gene), and at least 10 contiguous nucleotides of the sequenceshown in FIG. 1, wherein position 85146 is guanine, position 85161 isguanine, position 85216 is adenosine, position 85292 is cytosine, orposition 85300 is guanine; or an isolated nucleic acid moleculecomprising a polymorphic position selected from the group consisting ofpositions 35728, 36016, and 38867 of the nucleic acid sequence shown inFIG. 2 (SEQ ID NO: 2) (part of the DNAJC27 gene), and at least 10contiguous nucleotides of the sequence shown in FIG. 2, wherein position35728 is guanine, position 36016 is guanine, or position 38867 isguanine. It is recognized that SEQ ID NO: 1 is already known, and thenucleic acid molecule therefore does not encompass one that consists ofSEQ ID NO: 1.

Preferably, the nucleic acid molecule which comprises at least 15, morepreferably at least 20, still more preferably at least 25, contiguousbases of SEQ ID NO: 1 or SEQ ID NO: 2 adjacent to the polymorphic site.In one embodiment, the isolated nucleic acid molecule comprises not morethan 1,500 nt, preferably not more than 1000 nt, more preferably notmore than 900 nt, more preferably not more than 800 nt, more preferablynot more than 700 nt, preferably not more than 600 nt, more preferablynot more than 500 nt, preferably not more than 400 nt, more preferablynot more than 300 nt, more preferably not more than 150 nt., preferablynot more than 100 nt., still more preferably not more than 50 nt.

The nucleic acid molecule preferably contains the polymorphic site whichis within 4 nucleotides of the center of the nucleic acid molecule.Preferably, the polymorphic site is at the center of the nucleic acidmolecule.

In another embodiment, the nucleic acid molecule contains thepolymorphic site which is at the 3′-end of the nucleic acid molecule.

In another embodiment, the nucleic acid molecule contains thepolymorphic site which is at the 5′-end of the nucleic acid molecule.

The present invention also provides an array of nucleic acid moleculescomprising at least two nucleic acid molecules described above.

The present invention further provides a kit comprising a nucleic acidmolecule described above, and a suitable container.

Also provided is a method for detecting single nucleotide polymorphism(SNP) in the bovine DNAJC15 or DNAJC27 genes, wherein the DNAJC15 geneis partially shown in FIG. 1, and the DNAJC27 gene is partially shown inFIG. 2, the method comprising optionally isolating a nucleic acid samplefrom the bovine animal or a tissue sample therefrom, determining theidentity of a nucleotide of at least one position selected from thegroup consisting of a first polymorphic position comprising positions85146, 85161, 85216, 85292, and 85300 of SEQ ID NO: 1, and a secondpolymorphic position comprising positions 35728, 36016, and 38867 of SEQID NO: 2, and comparing the nucleotide identity of the positionrespectively to the nucleotide identity at a corresponding position ofSEQ ID NO: 1 or SEQ ID NO: 2.

Also provided is a method for genotyping a bovine cell, comprisingobtaining a nucleic acid sample from said cell and determining theidentity of a nucleotide of at least one position selected from thegroup consisting of positions 85146, 85161, 85216, 85292, and 85300 ofSEQ ID NO: 1, and positions 35728, 36016, and 38867 of SEQ ID NO: 2. Inone embodiment, the bovine cell is an adult cell, an embryo cell, asperm, an egg, a fertilized egg, or a zygote. In one embodiment, bothcopies of the gene in the cell are genotyped.

The present invention further provides a method for progeny testing ofcattle, the method comprising collecting a nucleic acid sample from saidprogeny, and genotyping said nucleic sample.

In another embodiment, a method is provided for selectively breedingcattle using a multiple ovulation and embryo transfer procedure (MOET),the method comprising superovulating a female animal, collecting eggsfrom said superovulated female, in vitro fertilizing said eggs from asuitable male animal, implanting said fertilized eggs into other femalesallowing for an embryo to develop, and genotyping said developingembryo, and terminating pregnancy if the developing embryo does not haveat least one polymorph selected from the group consisting of a firstpolymorph selected from the group consisting of guanine at position85146, guanine at position 85161, adenosine at position 85216, cytosineat position 85292, guanine at position 85300 of SEQ ID NO: 1; and asecond polymorph selected from the group consisting of guanine atposition 35728, guanine at position 36016, and guanine at position 38867of SEQ ID NO: 2.

In one embodiment, pregnancy is terminated if the developing embryo doesnot have either the first polymorph or the second polymorph.

In another embodiment, pregnancy is terminated if the developing embryois not homozygous in the first polymorph and the second polymorph.

Further provided is a method for selecting a cattle as a breeder,wherein the cattle is genotyped according to the present invention, andthe animal is only selected for breeding purpose if it comprises atleast a first polymorph or a second polymorph, wherein the firstpolymorph is selected from guanine at position 85146, guanine atposition 85161, adenosine at position 85216, cytosine at position 85292,guanine at position 85300 of SEQ ID NO: 1; and the second polymorph isselected from the group consisting of guanine at position 35728, guanineat position 36016, and guanine at position 38867 of SEQ ID NO: 2. In oneembodiment, the cattle animal is selected only if it comprises both thefirst and second polymorphs. In another embodiment, the cattle animal isselected only if it is homozygous with regard to both the first andsecond polymorphs.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a portion of the DNAJC15 gene sequence (SEQ ID NO: 1) wherethe polymorphic sites and some of the PCR primers are shown. The fivepolymorphic sites are highlighted, bold faced and underlined.

FIG. 2 shows a portion of the DNAJC27 gene sequence (SEQ ID NO: 2) wherethe polymorphic sites and some of the PCR primers are shown. The threepolymorphic sites are highlighted, bold faced and underlined.

FIG. 3 shows the changes in expression levels of heat shock proteingenes in bovine embryos using qRT-PCR. Data are shown as mean+/−maximumand minimum fold changes. Upregulation in degenerate or blastocystembryos is represented by bars above or below, respectively, the x axis.qRT-PCR was performed in 4 sets of biological replicates of blastocystsand degenerate embryos.

FIG. 4 shows the splice variants of DNAJC5, DNAJC19, DNAJC24, andDNAJB12 genes. Positions of start and stop codons are indicated byvertical arrows and positions of primers used in the qRT-PCR areindicted by horizontal arrows. Black boxes represent coding sequencesand white boxes represent untranslated regions. Due to overlap forDNAJB12, only transcript DNAB12-1220 could be amplified usingtranscript-specific primers.

FIG. 5 is a heat map of linkage disequilibrium (r²) between SNPs inDNAJC15 and DNAJC27. SNPs 85146, 85161, 85216, 85292 and 85300 arelocated in DNAJC15 while SNPs 35728, 36016 and 38867 are located inDNAJC27.

DETAILED DESCRIPTION OF THE INVENTION

The present inventor has constructed an in-vitro fertilization (IVF)system which has the advantages of a unified environment andwell-isolated components of the embryonic development process, and usedthis system to characterize genetic factors involved in embryonic loss,and hence infertility, and to associate candidate genes and pathwayswith fertilization rate and embryonic survival at both the genomic andgene expression levels (Khatib et al., 2008a,b; Khatib et al., 2009;Huang et al., 2010a,b).

As indicated above, HSPs are among the first proteins produced duringembryonic development (Neuer et al., 1999). The present inventorhypothesized that determining expression patterns of these genes indeveloped vs. arrested embryos could lead to the identification ofspecific genes involved in reproductive success. The high structural andfunctional conservation of HSPs during evolution suggests crucial rolesof these proteins in fertilization, embryo development, and thusfertility in cattle. Also, some HSPs are considered housekeeping genesthat are essential for many cell functions.

In efforts leading to the present invention, the expression levels ofHSP genes in biological replicate bovine embryo pools that differ intheir morphology and developmental statuses were evaluated, andassociations of polymorphisms with fertility traits were examined. TheHSP genes were found to be differentially expressed between blastocystsand degenerate embryos.

SNPs in two HSP genes were found to be associated with fertility traits.Specifically, a total of 17 candidate genes were chosen based ondifferential expression results observed in a previous microarrayanalysis of bovine embryos. To test whether or not these genes haveroles in early development of cattle embryos, their expression levelswere quantified and compared between blastocysts and degenerates. All 17genes showed expression differences that ranged between 1.5- and7.6-fold between the embryo groups. Two of these bovine HSP genes,DNAJC15 and DNAJC27, both of the HSP40 gene family, have severalpolymorphic positions, and these polymorphisms are associated withfertilization and blastocyst rates. Furthermore, all SNPs in each ofDNAJC15 and DNAJC27 genes showed high linkage disequilibrium with eachother.

The term “polymorphic” or “polymorphism” as used herein refers to theoccurrence of two or more alternative genomic sequences or allelesbetween or among different genomes or individuals. “Polymorphic” refersto the condition in which two or more variants of a specific genomicsequence can be found in a population. A “polymorphic site” is the locusat which the variation occurs. Polymorphisms generally have at least twoalleles, each occurring at a significant frequency in a selectedpopulation. A polymorphic locus may be as small as one base pair. Thefirst identified allelic form is arbitrarily designated as the referenceform, and other allelic forms are designated as alternative or variantalleles. The allelic form occurring most frequently in a selectedpopulation is sometimes referred to as the wild type form. Diploidorganisms may be homozygous or heterozygous for allelic forms. Abiallelic polymorphism has two forms, and a triallelic polymorphism hasthree forms, and so on.

Polymorphisms may provide functional differences in the geneticsequence, through changes in the encoded polypeptide, changes in mRNAstability, binding of transcriptional and translation factors to the DNAor RNA, and the like. Polymorphisms are also used to detect geneticlinkage to phenotypic variation.

One type of polymorphism, single nucleotide polymorphisms (SNPs), hasgained wide use for the detection of genetic linkage recently. SNPs aregenerally biallelic systems, that is, there are two alleles that anindividual may have for any particular SNP marker. In the instant case,the SNPs are used for determining the genotypes of the two HSP genes,which are found to have strong correlation to fertilization rate andembryonic survival.

The provided sequences also encompass the complementary sequencecorresponding to any of the provided polymorphisms. In order to providean unambiguous identification of the specific site of a polymorphism,the numbering of the original sequence in the GenBank is shown in FIG. 1and is used throughout this disclosure.

The present invention provides nucleic acid based genetic markers foridentifying bovine animals with superior breeding (such as fertility andembryo survival rates) traits. In general, for use as markers, nucleicacid fragments, preferably DNA fragments, may be as short as 7nucleotides (nt), but may preferably be at least 12 nt, 15 nt, usuallyat least 20 nt, often at least 50 nt. Such small DNA fragments areuseful as primers for the polymerase chain reaction (PCR), and/or probesfor hybridization screening, etc.

The term primer refers to a single-stranded oligonucleotide capable ofacting as a point of initiation of template-directed DNA synthesis underappropriate conditions (i.e., in the presence of four differentnucleoside triphosphates and an agent for polymerization, such as, DNAor RNA polymerase or reverse transcriptase) in an appropriate buffer andat a suitable temperature. The appropriate length of a primer depends onthe intended use of the primer but typically ranges from 15 to 30nucleotides. Short primer molecules generally require coolertemperatures to form sufficiently stable hybrid complexes with thetemplate. A primer need not reflect the exact sequence of the templatebut must be sufficiently complementary to hybridize with the template.The term primer site, or priming site, refers to the area of the targetDNA to which a primer hybridizes. The term primer pair means a set ofprimers including a 5′ upstream primer that hybridizes with the 5′ endof the DNA sequence to be amplified and a 3′, downstream primer thathybridizes with the complement of the 3′ end of the sequence to beamplified.

The term “probe” or “hybridization probe” denotes a defined nucleic acidsegment (or nucleotide analog segment) which can be used to identify byhybridizing to a specific polynucleotide sequence present in samples,said nucleic acid segment comprising a nucleotide sequence complementaryof the specific polynucleotide sequence to be identified. “Probes” or“hybridization probes” are nucleic acids capable of binding in abase-specific manner to a complementary strand of nucleic acid.

An objective of the present invention is SNP genotyping, that is, todetermine which embodiment of the SNP polymorphisms a specific sample ofDNA has. For example, it is desirable to determine whether thenucleotide at a particular position is A or C. Many references describegenotyping methods, such as Chen et al., “Single nucleotide polymorphismgenotyping: biochemistry, protocol, cost and throughput”,Pharmacogenomics J. 2003; 3(2):77-96; Kwok et al., “Detection of singlenucleotide polymorphisms”, Curr Issues Mol Biol. April 2003; 5(2):43-60;Shi, “Technologies for individual genotyping: detection of geneticpolymorphisms in drug targets and disease genes”, Am J Pharmacogenomics.2002; 2(3):197-205; and Kwok, “Methods for genotyping single nucleotidepolymorphisms”, Annu Rev Genomics Hum Genet 2001; 2:235-58. Exemplarytechniques for high-throughput SNP genotyping are described inMarnellos, “High-throughput SNP analysis for genetic associationstudies”, Curr Opin Drug Discov Devel. May 2003; 6(3):317-21. Common SNPgenotyping methods include, but are not limited to, TaqMan assays andmodifications thereof such as Molecular Beacon assays, SNPlex platforms,Bio-Plex system, CEQ and SNPstream systems, Molecular Inversion Probearray technology, BeadArray Technologies (e.g., Illumina GoldenGate andInfinium assays), single stranded conformation polymorphism assays(SSCP), molecular beacon assays, nucleic acid arrays, allele-specificprimer extension, allele-specific PCR, arrayed primer extension,homogeneous primer extension assays, primer extension with detection bymass spectrometry, pyrosequencing, multiplex primer extension sorted ongenetic arrays, ligation with rolling circle amplification, homogeneousligation, OLA (U.S. Pat. No. 4,988,167), multiplex ligation reactionsorted on genetic arrays, restriction-fragment length polymorphism,single base extension-tag assays, and the Invader assay. Such methodsmay be used in combination with detection mechanisms such as, forexample, luminescence or chemiluminescence detection, fluorescencedetection, time-resolved fluorescence detection, fluorescence resonanceenergy transfer, fluorescence polarization, mass spectrometry, andelectrical detection.

The sequence neighboring the SNP site can be used to design SNPdetection reagents such as oligonucleotide probes, which may optionallybe implemented in a kit format. Preferably, the oligonucleotide probewill have a detectable label, and contains for example an A at thecorresponding position. Experimental conditions can be chosen such thatif the sample DNA contains an A, a hybridization signal can be detectedbecause the probe hybridizes to the corresponding complementary DNAstrand in the sample, while if the sample DNA contains a G, nohybridization signal is detected.

Similarly, PCR primers and conditions can be devised, whereby theoligonucleotide is used as one of the PCR primers, for analyzing nucleicacids for the presence of a specific sequence. These may be directamplification of the genomic DNA, or RT-PCR amplification of the mRNAtranscript of the target gene. The use of the polymerase chain reactionis described in Saiki et al. (1985) Science 230:1350-1354. Amplificationmay be used to determine whether a polymorphism is present, by using aprimer that is specific for the polymorphism. Alternatively, variousmethods are known in the art that utilize oligonucleotide ligation as ameans of detecting polymorphisms, for examples see Riley et al (1990)Nucleic Acids Res. 18:2887-2890; and Delahunty et al (1996) Am. J. Hum.Genet. 58:1239-1246. The detection method may also be based on directDNA sequencing, or hybridization, or a combination thereof. Where largeamounts of DNA are available, genomic DNA is used directly.Alternatively, the region of interest is cloned into a suitable vectorand grown in sufficient quantity for analysis. The nucleic acid may beamplified by PCR, to provide sufficient amounts for analysis.

Hybridization may be performed in solution, or such hybridization may beperformed when either the oligonucleotide probe or the targetpolynucleotide is covalently or noncovalently affixed to a solidsupport. Attachment may be mediated, for example, by antibody-antigeninteractions, poly-L-Lys, streptavidin or avidin-biotin, salt bridges,hydrophobic interactions, chemical linkages, UV cross-linking baking,etc. Oligonucleotides may be synthesized directly on the solid supportor attached to the solid support subsequent to synthesis. Solid-supportssuitable for use in detection methods of the invention includesubstrates made of silicon, glass, plastic, paper and the like, whichmay be formed, for example, into wells (as in 96-well plates), slides,sheets, membranes, fibers, chips, dishes, and beads. The solid supportmay be treated, coated or derivatized to facilitate the immobilizationof the allele-specific oligonucleotide or target nucleic acid. Forscreening purposes, hybridization probes of the polymorphic sequencesmay be used where both forms are present, either in separate reactions,spatially separated on a solid phase matrix, or labeled such that theycan be distinguished from each other.

Hybridization may also be performed with nucleic acid arrays andsubarrays such as described in WO 95/11995. The arrays would contain abattery of allele-specific oligonucleotides representing each of thepolymorphic sites. One or both polymorphic forms may be present in thearray, for example the polymorphism of a SNP position may be representedby either, or both, of the listed nucleotides. Usually such an arraywill include at least 2 different polymorphic sequences, i.e.polymorphisms located at unique positions within the locus, and mayinclude all of the provided polymorphisms. Arrays of interest mayfurther comprise sequences, including polymorphisms, of other geneticsequences, particularly other sequences of interest. The oligonucleotidesequence on the array will usually be at least about 12 nt in length,may be the length of the provided polymorphic sequences, or may extendinto the flanking regions to generate fragments of 100 to 200 nt inlength. For examples of arrays, see Ramsay (1998) Nat. Biotech. 16:4044;Hacia et al. (1996) Nature Genetics 14:441-447; Lockhart et al. (1 996)Nature Biotechnol. 14:1675-1680; and De Risi et al. (1996) NatureGenetics 14:457-460.

The identity of polymorphisms may also be determined using a mismatchdetection technique, including but not limited to the RNase protectionmethod using riboprobes (Winter et al., Proc. Natl. Acad. Sci. USA82:7575, 1985; Meyers et al., Science 230:1242, 1985) and proteins whichrecognize nucleotide mismatches, such as the E. coli mutS protein(Modrich, P. Ann. Rev. Genet. 25:229-253, 1991). Alternatively, variantalleles can be identified by single strand conformation polymorphism(SSCP) analysis discussed above (Orita et al., Genomics 5:874-879, 1989;Humphries et al., in Molecular Diagnosis of Genetic Diseases, R. Elles,ed., pp. 321-340, 1996) or denaturing gradient gel electrophoresis(DGGE) (Wartell et al., Nucl. Acids Res. 18:2699-2706, 1990; Sheffieldet al., Proc. Natl. Acad. Sci. USA 86:232-236, 1989).

A polymerase-mediated primer extension method may also be used toidentify the polymorphism(s). Several such methods have been describedin the patent and scientific literature and include the “Genetic BitAnalysis” method (WO92/15712) and the ligase/polymerase mediated geneticbit analysis (U.S. Pat. No. 5,679,524). Related methods are disclosed inWO91/02087, WO90/09455, WO95/17676, U.S. Pat. Nos. 5,302,509, and5,945,283. Extended primers containing a polymorphism may be detected bymass spectrometry as described in U.S. Pat. No. 5,605,798. Anotherprimer extension method is allele-specific PCR (Ruao et al., Nucl. AcidsRes. 17:8392, 1989; Ruao et al., Nucl. Acids Res. 19, 6877-6882, 1991;WO 93/22456; Turki et al., J. Clin. Invest. 95:1635-1641, 1995). Inaddition, multiple polymorphic sites may be investigated bysimultaneously amplifying multiple regions of the nucleic acid usingsets of allele-specific primers as described in Wallace et al. (WO89/10414).

A detectable label may be included in an amplification reaction.Suitable labels include fluorochromes, e.g. fluorescein isothiocyanate(FITC), rhodamine, Texas Red, phycoerythrin, allophycocyanin,6-carboxyfluorescein (6-FAM),2′,7′-dimethoxy-4′,5′-dichloro-6-carboxyfluorescein (JOE),6-carboxy-X-rhodamine (ROX),6-carboxy-2′,4′,7′,4,7-hexachlorofluorescein (HEX), 5-carboxyfluorescein(5-FAM) or N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA), radioactivelabels, e.g. ³²P, ³⁵S, ³H; etc. The label may be a two stage system,where the amplified DNA is conjugated to biotin, haptens, etc. having ahigh affinity binding partner, e.g. avidin, specific antibodies, etc.,where the binding partner is conjugated to a detectable label. The labelmay be conjugated to one or both of the primers. Alternatively, the poolof nucleotides used in the amplification is labeled, so as toincorporate the label into the amplification product.

It is readily recognized by those ordinarily skilled in the art that inorder to maximize the signal to noise ratio, in probe hybridizationdetection procedure, the polymorphic site should at the center of theprobe fragment used, whereby a mismatch has a maximum effect ondestabilizing the hybrid molecule; and in a PCR detection procedure, thepolymorphic site should be placed at the very 3′-end of the primer,whereby a mismatch has the maximum effect on preventing a chainelongation reaction by the DNA polymerase. The location of nucleotidesin a polynucleotide with respect to the center of the polynucleotide aredescribed herein in the following manner. When a polynucleotide has anodd number of nucleotides, the nucleotide at an equal distance from the3′ and 5′ ends of the polynucleotide is considered to be “at the center”of the polynucleotide, and any nucleotide immediately adjacent to thenucleotide at the center, or the nucleotide at the center itself isconsidered to be “within 1 nucleotide of the center.” With an odd numberof nucleotides in a polynucleotide any of the five nucleotides positionsin the middle of the polynucleotide would be considered to be within 2nucleotides of the center, and so on. When a polynucleotide has an evennumber of nucleotides, there would be a bond and not a nucleotide at thecenter of the polynucleotide. Thus, either of the two centralnucleotides would be considered to be “within 1 nucleotide of thecenter” and any of the four nucleotides in the middle of thepolynucleotide would be considered to be “within 2 nucleotides of thecenter,” and so on.

In some embodiments, a composition contains two or more differentlylabeled oligonucleotides for simultaneously probing the identity ofnucleotides or nucleotide pairs at two or more polymorphic sites. It isalso contemplated that primer compositions may contain two or more setsof allele-specific primer pairs to allow simultaneous targeting andamplification of two or more regions containing a polymorphic site.

Alternatively, the relevant portion of the target gene of the sample ofinterest may be amplified via PCR and directly sequenced, and thesequence be compared to the wild type sequence. It is readily recognizedthat, other than those specifically disclosed herein, numerous primerscan be devised to achieve the objectives. PCR and sequencing techniquesare well known in the art and reagents and equipments are readilyavailable commercially.

The so-called next generation sequencing, and high-throughput sequencingmethods, may also be sued. For example, Massively Parallel SignatureSequencing (MPSS); Polony sequencing, pyro sequencing, SOLiD sequencing;ion semiconductor sequencing, and DNA nanoball sequencing systems arewell-known to those skilled in the art.

DNA markers have several advantages; segregation is easy to measure andis unambiguous, and DNA markers are co-dominant, i.e., heterozygous andhomozygous animals can be distinctively identified. Once a marker systemis established selection decisions could be made very easily, since DNAmarkers can be assayed any time after a sample is collected from theindividual animal, or even earlier by testing embryos in vitro if veryearly embryos are collected. The use of marker assisted geneticselection will greatly facilitate and speed up cattle breeding problems.For example, a modification of the multiple ovulation and embryotransfer (MOET) procedure can be used with genetic marker technology.Specifically, females are superovulated, eggs are collected, in vitrofertilized using semen from superior males and implanted into otherfemales allowing for use of the superior genetics of the female (as wellas the male) without having to wait for her to give birth to one calf ata time. Developing blastomeres at the 4-8 cell stage may be assayed forpresence of the marker, and selection decisions made accordingly.

In one embodiment of the invention an assay is provided for detection ofpresence of a desirable genotype using the markers.

The term “genotype” as used herein refers to the identity of the allelespresent in an individual or a sample. In the context of the presentinvention a genotype preferably refers to the description of thepolymorphic alleles present in an individual or a sample. The term“genotyping” a sample or an individual for a polymorphic marker refersto determining the specific allele or the specific nucleotide carried byan individual at a polymorphic marker.

The present invention is suitable for identifying a bovine, including ayoung or adult bovine animal, an embryo, a semen sample, an egg, afertilized egg, or a zygote, or other cell or tissue sample therefrom,to determine whether said bovine possesses the desired genotypes of thepresent invention, some of which are indicative of improvedfertilization rate and embryonic survival.

Further provided is a method for genotyping the bovine HSP gene,comprising determining for the two copies of the HSP gene present theidentity of the nucleotide pair at positions 25402 and 19069.

One embodiment of a genotyping method of the invention involvesexamining both copies of the relevant HSP gene, or a fragment thereof,to identify the nucleotide pair at the polymorphic site in the twocopies to assign a genotype to the individual. In some embodiments,“examining a gene” may include examining one or more of: DNA containingthe gene, mRNA transcripts thereof, or cDNA copies thereof. As will bereadily understood by the skilled artisan, the two “copies” of a gene,mRNA or cDNA, or fragment thereof in an individual may be the sameallele or may be different alleles. In another embodiment, a genotypingmethod of the invention comprises determining the identity of thenucleotide pair at the polymorphic site.

The present invention further provides a kit for genotyping a bovinesample, the kit comprising in a container a nucleic acid molecule, asdescribed above, designed for detecting the polymorphism, and optionallyat least another component for carrying out such detection. Preferably,a kit comprises at least two oligonucleotides packaged in the same orseparate containers. The kit may also contain other components such ashybridization buffer (where the oligonucleotides are to be used as aprobe) packaged in a separate container. Alternatively, where theoligonucleotides are to be used to amplify a target region, the kit maycontain, preferably packaged in separate containers, a polymerase and areaction buffer optimized for primer extension mediated by thepolymerase, such as PCR.

In one embodiment the present invention provides a breeding methodwhereby genotyping as described above is conducted on bovine embryos,and based on the results, certain cattle are either selected or droppedout of the breeding program.

Through use of the linked marker loci, procedures termed “markerassisted selection” (MAS) may be used for genetic improvement within abreeding nucleus; or “marker assisted introgression” for transferringuseful alleles from a resource population to a breeding nucleus (Soller1990; Soller 1994).

Testing of Roles of 17 HSP Genes

The roles of 17 HSP genes in early embryonic development and fertilityin cattle at both genomic and gene expression levels were investigated.These genes include 8 HSP40 genes, six HSP70 genes, one HSPP, and 2HSP10 genes. (see Table 1) These genes were chosen based on differentialexpression results observed in a previous microarray analysis of bovineembryos; and their expression levels were quantified and comparedbetween blastocysts and degenerates to test whether or not these geneshave roles in early development of cattle embryos.

All 17 genes showed expression differences that ranged between 1.5- and7.6-fold between the embryo groups. All HSP40 family genes were found tobe upregulated in degenerate embryos compared to blastocysts. Althoughthe specific functions of HSP40s have not been reported in cattle,studies from other species have shown that these genes play importantroles in protecting cells under stress conditions (Lanneau et al.,2007). It is well established that ATP hydrolysis is necessary forprotein folding activity of HSP70s and that HSP40s stimulate ATPaseactivity and stabilize the interaction of HSP70s with their substrates(Qiu et al., 2006). Gotoh and colleagues (2004) reported that HSP40combines with HSP70 to act as chaperones to protect cells fromapoptosis. Degenerate embryos in the IVF system are growth-arrestedembryos and seem to undergo partial apoptosis. Research has shown thatHSPs are upregulated to prevent apoptosis triggered by different stimuliby interacting with key factors of the apoptotic signaling pathways(Lanneau et al., 2007). Therefore, it is feasible that upregulation ofHSP40 genes observed in degenerates is a response to stress on theseembryos so they can maintain protein homeostasis.

The qRT-PCR analysis revealed moderate fold change expression of HSPE1,HSPH1, HSB1, and six HSP70 genes (HSPA2, HSPA4, HSPA5, HSPA8, HSPA9 andHSPA14). HSPA2, HSPA4, HSPA5, and HSPA8 showed higher expression inblastocysts than degenerates, whereas expression levels of HSPA9 andHSPA14 were higher in degenerate embryos. Although the specificfunctions of most HSP70s have not been reported in cattle, studies inhuman and mouse have indicated important roles for these genes in earlyembryogenesis. It has been reported that Hspa2 is essential for normalspermatogenesis and for growth and survival of cancer cells and thatHspa8 knockout mice are not viable because of the housekeeping functionsof this gene (Daugaard et al., 2007). Human HSPA5 is involved in thefolding and transport of proteins into the endoplasmic reticulum andHspa5 knockout mice embryos die at day 3.5, and therefore it, too, isconsidered a housekeeping gene (Daugaard et al., 2007). Furthermore,Matwee et al. (2001) have reported a reduced blastocyst rate and anincreased apoptosis of embryos cultured in the presence of anti-HSP70.Thus, upregulation and downregulation of HSPs in bovine embryos observedin the present study imply that these genes play vital roles in earlyembryo development.

Given that alternative splicing is a major source of phenotypiccomplexity in mammals (Wang et al., 2008), a search was conducted of theEnsembl annotation of the bovine genome and found that only 4 out of 17HSP candidate genes have annotated splice variants. In contrast, for the17 human and mouse counterparts, 15 and 13 genes, respectively, have 2or more splice variants. The low number of alternative transcriptsidentified in cattle is presumably due to the incomplete annotation ofthe bovine genome. Indeed, in a previous study, thousands of alternativesplicing events were characterized in blastocysts and degenerativeembryos, and some of those events were found to be associated with thedevelopmental status of embryos (Huang and Khatib, 2010).

In order to explore the effects of the different splice variants of HSPson embryo development, expression levels of the identified variants wereestimated and compared between blastocysts and degenerate embryos. Theresults clearly show that different splice variants display differentexpression levels which imply different functions in embryo development.For example, while the long variant of DNAJC24 was highly expressed indegenerate embryos compared to blastocysts, the short variant of thisgene was found to be lowly expressed in both embryo groups. Also, forDNAJC5, one transcript could not be detected using qRT-PCR. Theseresults are consistent with human studies in which only some of thesplice variants of genes were found to be associated with a diseasestate (Wang and Cooper, 2007). Collectively, these results testify tothe importance of alternative splicing as a key regulator of phenotypicvariation in mammals.

To better understand the involvement of the HSP genes in fertilitytraits, 4 genes that showed the most significant expression differencesbetween embryo groups were tested for SNP association.

Recently, an IVF experimental system in cattle has been developed in ourlaboratory (see Methods) aimed at identifying genetic factors affectingfertilization and embryo development. The effectiveness of this systemin identifying genes and pathways associated with developmental andfertility traits has been demonstrated in several studies (Khatib etal., 2008a,b; Khatib et al., 2009; Driver et al., 2009; Wang et al.,2009; Huang et al., 2010a).

Analysis of DNAJC15 and DNAJC27 SNP revealed significant associationswith blastocyst and fertilization rates, respectively. These results areof particular significance because these 2 genes showed a remarkabledifference in expression between blastocysts and degenerate embryos,providing strong evidence for the involvement of these genes in embryoproduction and development in cattle. The results also provide furtherevidence for the roles of HSPs in the fertilization process andblastocyst rates observed in mice and cattle in previous studies. Neueret al. (1998) found that the presence of anti-HSP60 monoclonalantibodies had significant effects on blastocyst rates of mouse embryos.Matwee et al. (2001) reported that the addition of anti-HSP70 monoclonalantibodies to the culture medium reduced the mean number of spermatozoabound to zona pellucida by about 50% to that of controls. Also, theauthors reported that anti-HSP70 significantly reduced the number ofbovine embryos that reached the blastocyst stage (Matwee et al., 2001).A recent study by Rosenkrans et al. (2010) reported an associationbetween SNP in the promoter region of HSP70 and calving percentages inBrahman cows. Although a small sample size was used in the associationanalysis, the results point to an important role of HSP70 in cowfertility.

The identification of specific HSP genes contributing to embryo survivalcan be a unique opportunity to improve protection of IVF embryos fromdifferent toxic conditions and to enhance pregnancy rates in cattle(Hansen, 2007). HSP genes investigated in this study were found to bedifferentially expressed between blastocysts and degenerate embryos andSNP in 2 genes were associated with fertility traits. Blastocysts anddegenerate embryos have distinct morphological and developmentalfeatures. As such, the present study provides a set of candidate geneticmarkers for pre-implantation embryo development. However, it has beenreported that relatively small changes (decrease or increase) inexpression levels of heat shock proteins can result in growthabnormalities and cell death (Nollen and Morimoto, 2002). Also, theassessment of the expression of alternative transcript isoforms in theembryos examined in this study testifies to the importance of inclusionof this approach in studying gene expression.

The present invention accordingly provides novel cattle genotyping,selective cattle breeding and related methods, based on the discoverythat certain HSP40 polymorphism confers improved fertilization andembryonic survival rate to cattle.

The following examples are intended to illustrate preferred embodimentsof the invention and should not be interpreted to limit the scope of theinvention as defined in the claims.

EXAMPLES Materials and Methods

In-Vitro Maturation, Fertilization, and Embryo Culture

In this study, fertilization and embryo production were performed in 2different experiments. The first experiment is a comparison ofexpression profiles of 17 HSPs between 2 populations of embryosdiffering in their morphology, and the second experiment is a genomicassociation analysis of genes—found to be differentially-expressed inthe first experiment—with fertilization and blastocyst rates.

Experiment I: Expression Analysis of HSP Genes in Cattle Embryos

Ovaries were obtained from a local abattoir and processed for IVF usingstandard protocol (Huang et al., 2010b; Khatib et al., 2008a). Briefly,oocytes were aspirated from 2-6 mm follicles and washed in Tyrode'salbumin lactate pyruvate (TALP)-Hepes after which they underwentmaturation in supplemented M199 media (Khatib et al., 2008a). Incubationwas completed at 5% CO₂ in air at 39° C. and high humidity for 22-24hours at which time oocytes were then washed in TALP-Hepes and movedinto fresh IVF-TALP (Biowhittaker, Walkersburg, Md.). Semen samples from2 different bulls were used for fertilization; sperm underwent Percollseparation (45-90% gradient) and were adjusted to a final concentrationof 1×10⁶ /ml (Parrish et al., 1995). Fertilization was marked as day 0of development and completed by combining sperm, heparin, and PHE withthe oocytes in 44 μl drops of media (Khatib et al., 2008a). Incubationwas then continued for 22-24 hours after which the putative zygotes werewashed in TALP-Hepes, denuded of remaining cumulus cell complexes, andplaced into supplemented synthetic oviductal fluid (Biowhittaker) andreturned to incubation (Khatib et al., 2008a).

Morphological Assessment

Putative zygotes were cultured over an 8-day period during whichassessments of their developmental progress were made. By day 5 ofdevelopment, a bovine embryo should attain approximately 16-32 cells andshow signs of cellular compaction deeming it as morula. Embryos failingto show these characteristics were assumed to have been arrested indevelopment and were excluded from analysis. Morulas were then continuedin culture until day 8. By this time an embryo should show evidence of afluid filled cavity (blastocoele), which gives rise to thedifferentiation of the inner cell mass and the trophectoderm, qualifyingit as a blastocyst. For expression analysis, there were 2 populations ofembryos collected. The first consisted of embryos that attained acompacted morula status by day 5 but failed to form a blastocoele by day8, referred as degenerate embryos. The second consisted of embryos thatdeveloped into blastocysts by day 8. Embryos from each morphologicalgroup were collected in pools of 20 and preserved in RNALater (Ambion,Austin, Tex.). Two bulls were used with 2 sets of biological replicatepools (total of 4 biological replicates) from each to prevent maternalcrossover.

Quantitative Real-Time PCR (qRT-PCR)

A previous study in our laboratory using microarray expression analysishas revealed that many genes are differentially expressed betweenblastocysts and degenerate embryos (Huang et al., 2010b). Seventeen HSPgenes that showed 1.5-fold or higher differences in expression betweenthe embryo groups described in the study of Huang et al. (2010b) werechosen for validation of differential expression and furtherinvestigation in new sets of blastocysts and degenerate embryo poolsusing qRT-PCR analysis. Primers (Table 1) were designed to amplifyfragments spanning more than one exon to exclude the possibility ofgenomic DNA contamination in the qRT-PCR reactions using the BeaconDesigner software (Premier Biosoft International, Palo Alto, Calif.).Total RNA was extracted from pools of embryos using RNaqueous Micro(Ambion) and quality controlled using a RNA6000 PicoChip (AgilentTechnologies, Calif.). Messenger RNA was amplified using MessageAmp II(Ambion, Austin, Tex.), followed by cDNA synthesis using iScript(Bio-Rad, Hercules, Calif.) according to manufacturers' instructions.Reactions of qRT-PCR were run on a DNA Engine-Opticon 2 Detection System(MJ Research, Watertown, Mass.) using iQ SYBR Green Supermix kit(Bio-Rad Laboratories, Calif.). Each sample was run in quadruplicate andall 4 expression results were averaged. Analysis of gene expressionlevels was conducted using the 2^(−ΔΔCt) method (Livak and Schmittgen,2001). The selection of the housekeeping gene GAPDH as an endogenouscontrol was as described in Huang et al. (2010b).

Experiment II: Association Analysis of Differentially-Expressed HSPswith Fertilization and Blastocyst Rates

In order to further investigate the involvement in fertility of genes(DNAJC15, DNAJC19, DNAJC24, and DNAJC27) that showed the highest foldchange in expression in degenerate embryos vs. blastocysts, anassociation analysis between SNPs in these genes and 2 main fertilitytraits: fertilization rate and blastocyst rate was performed. Using theIVF experimental system, several genes were found to be significantlyassociated with variation in fertilization and embryonic survival rates(Khatib et al., 2008a,b; Khatib et al., 2009).

Phenotypic Data. In order to generate phenotypic data for associationanalysis, a total of 6,893 in-vitro fertilizations were performed usingoocytes from 399 ovaries (obtained from 399 Holstein cows) and semensamples from 12 Holstein bulls. For 92 ovaries, oocytes were fertilizedby two different bulls each. Fertilization rate was calculated as thenumber of cleaved embryos 48 h postfertilization out of the total numberof oocytes exposed to sperm. Blastocyst rate was calculated as thenumber of embryos that reached blastocyst stage out of the total embryoscultured by day 8. asdf

Genotyping. DNA was extracted from ovaries (n=399) using standardphenol/chloroform protocols. The DNA concentrations were measured usinga spectrophotometer (Ultraspec 2100; Amersham Biosciences). Forpolymorphism identification, 3 DNA pools were constructed from 20different ovary/cow samples to contain 25 ng of DNA from each sample.DNA Pools were amplified with different sets of primers designed fromthe coding and 5′ and 3′ UTR regions of the 4 candidate genes (Table 3).Amplification, sequencing of PCR products, and SNP identification wereas described in Khatib et al. (2008a,b). SNP identified in DNAJC15,DNAJC19, DNAJC24, and DNAJC27 were genotyped for the 399 ovary/cowsamples at GeneSeek (Lincoln, Nebr.).

Statistical Analysis

For expression analysis, normalized gene expression values (ΔCt) wereanalyzed using a general linear model including the fixed effects of thebull, the type of embryo (blastocyst or degenerate), and the randomeffect of the pool. Association between the normalized gene expressionand the type of embryo was tested using a likelihood ratio test bycomparing this model to a reduced model without the embryo effect. Themean and the range of the fold change for each gene were calculated as2^(−ΔΔCt) using the estimated ΔΔCt value±standard error.

For genomic association analysis between SNP and fertilization andblastocyst rates, the following mixed linear model was used,

y _(ijk) =μ+o _(i) +b _(j)SNP_(ijk) +e _(ijk)

where y_(ijk) represents in turn, the fertilization or survival rate ofoocytes k from ovary i fertilized with semen from bull j; μ represents ageneral mean for the trait considered, o_(i) represents the randomeffect of the individual ovary from which oocytes were harvested; b_(j)represents the random effect of the sire used in the fertilization;SNP_(ijk) represents the fixed effect of the genotypic class for the SNPconsidered; and e_(ijk) represents the residuals, assumed normal,independent and identically distributed with mean 0 an variance Iσ_(e)². Ovaries and bulls were assumed uncorrelated with variance structuresIσ_(o) ² and I_(b) ², respectively. Association between the SNP andfertilization or blastocyst rates was tested using a likelihood ratiotest by comparing the full model to a reduced model without the SNPeffect. All analyses were performed using the lme4 package of Rlanguage/environment (R Development Core Team, 2009).

Results

Expression Profiling of HSP Genes in Embryos

Expression differences between degenerate embryos and blastocysts wereestimated for 17 HSP genes in 4 sets of biological replicates usingqRT-PCR (FIG. 1). A range of 1.5- to 7.6-fold difference in expressionwas observed between embryo groups. Interestingly, all HSP40 gene familymembers were found to be upregulated in degenerate embryos compared toblastocysts (FIG. 1). For example, DNAJC15 (P<0.0001), DNAJC19(P<0.0001), DNAJC24 (P=0.002), and DNAJC27 (P=0.0098) showed an averageof 7.6-, 4.8-, 3.3-, 4.33-fold differences in expression, respectively.In contrast, only 2 members of HSP70 family (HSPA14 and HSPA9) showedupregulation in degenerates, whereas 4 HSP70 genes (HSPA2, HSPA4, HSPA5,and HSPA8) showed higher expression levels in blastocysts than indegenerates (FIG. 1).

Differential Expression of Alternatively-Spliced Transcripts

Splice variants of HSP genes were identified using the genebuildprocedure of Ensembl (http://uswest.ensembl.org/). In this procedure,annotation of transcripts is based on mRNA and protein sequencesdeposited in public scientific databases. Ensembl search revealed onetranscript per gene for 13 genes, 2 transcripts each for DNAJB12 andDNAJC19, and 3 transcripts each for DNAJC5 and DNAJC24. Expressionlevels of splice variants of DNAJC5, DNAJC19, DNAJC24, and DNAJB12 (FIG.2) were estimated in 4 sets of biological replicates of blastocysts anddegenerate embryos using qRT-PCR. Transcripts DNAJC5-1049 and DNAJC5-667(FIG. 2) showed 2.53- and 1.67-fold higher expression, respectively, indegenerate embryos compared to blastocysts while the DNAJC5-725transcript was not detected in any of the embryo groups. For DNAJC19,only transcript DNAJC19-536 was detected in embryos. For DNAJC24,DNAJC24-2172 transcript showed a 3.22-fold higher expression indegenerate embryos compared to blastocysts, whereas transcriptDNAJC24-628 was lowly expressed in all embryo samples. DNAJC24-1893transcript could not be detected in embryos. The sequences of the 2DNAJB12 transcripts were overlapping so that only transcript DNAB12-1220could be amplified using transcript-specific primers. DNAB12-1220 showeda 1.95-fold higher expression in degenerate embryos vs. blastocysts.

Association of HSP Polymorphisms with Fertilization and Blastocyst Rates

SNP in genes DNAJC15, DNAJC19, DNAJC24, and DNAJC27 were tested forassociation with fertilization and blastocyst rates. These genes showedthe highest fold differences in expression between embryo groups(FIG. 1) and the highest statistical significance in the general linearmodel analysis (P<0.01). Using the pooled DNA sequencing approach, 5 SNPwere identified in the 3′UTR of DNAJC15, one SNP in exon 5 of DNAJC19,one SNP and one 4-bp deletion in the 3′ UTR of DNAJC24, and 3 SNP in the3′ UTR of DNAJC27. All SNP in each of DNAJC15 and DNAJC27 genes showedhigh linkage disequilibrium with each other (FIG. 3). Polymorphisms inDNAJC19 and DNAJC24 did not show significant associations with fertilitytraits. Table 2 shows fertilization and blastocysts rates for thegenotypic classes of the DNAJC15 and DNAJC27 SNP. For DNAJC27 SNP36016,oocytes collected from genotype GG ovaries showed a 69.3% fertilizationrate vs. 62.2% for oocytes collected from CC ovaries (P=0.034). ForDNAJC15 SNP85146, the blastocyst rate of embryos produced from GG damswas 40.1% vs. 31.0% and 28.1% for embryos produced from AA and AG dams,respectively (Table 2).

While the invention has been described in connection with one or moreembodiments, it should be understood that the invention is not limitedto those embodiments, and the description is intended to cover allalternatives, modifications, and equivalents, as may be included withinthe spirit and scope of the appended claims. All referenced cited hereinare further incorporated by reference in their entirety.

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TABLE 1Primers for total expression and alternative splicing analyses of HSP genesamplicon Gene Family forward primer 5′-3′ reverse primer 5′-3′ size (bp)GAPDH TGCCCAGAATATCATCCC AGGTCAGATCCACAACAG 134 DNAJC5 HSP40CTACGACAAGTACGGCTCAC GGCAGCAGCAACAGTAGC 149 DNAJC15 HSP40AGGTCGCTACGCATTTCAG GACTTGCTTCTCGCCTACTC 137 DNAJC19 HSP40GGACTGACCATTGCTGCTG CAAACCCACCTCTGTAATAGC 140 DNAJC24 HSP40GAAATATGGGACCAGTAGATGC TGTAACTTCTTCTGCTTCATCC 134 DNAJC27 HSP40AACAAGCGGACACCATTCG TGAAGCAGCACAGCAAGC 124 HSPA5 HSP70CAACCAACTGTTACCATCAAGG AAAGGTGACTTCAATCTGTGG 133 HSPA9 HSP70GACCAACTGCCTGCTGATG GATGCCGCCTGCCTTATG 113 HSPA8 HSC70CGCAGAAGCCTACCTTGG GTTGAGACCAGCAATAGTTCC 115 HSPA14 HSP70AACCTTAGCACAGTACCTAGC TGTCAGCACCGTTCATCAG 101 HSBP1 HSBPCATGTCCGACCAGATCATTG TTCACTGTCCAGCTCTTCC 115 HSPE1 HSP10GCAAGCAACGGTGGTAGC ACTTTGGTGCCTCCATATTCTG 117 DNAJB1 HSP40GAGGAGAAGTTCAAGGAGATCG TTAGTACCGCCGCTGCTC 134 DNAJB12 HSP40GCAAACTAGCCCTCAAATTCC CCTTGTCATCACCGAACTGG 141 DNAJC14 HSP40GTGAATGAGTTTCTGTCCAAGC GTATCTGGCACTCTTAGGTTCC 114 HSPA2 HSP70ACGCTGTGGAGTCCTATACC TTCCGCCATCTGGTTCCG 146 HSPA4 HSP70TCCTGCCTTAGAAGAGAAACC CCCAGTGTTGTGTCAAATGC 132 HSPH1 HSP100ATGTTGAGTTGCCTATTGAAGC CCTCCACCGCATTCTTAGC 141 Transcript DNAJC5-667AGGAGACGGAGTTCTATG CACGTTCACACCTCAAC 126 DNAJC5-725 GGCCCTGTTCATCTTCTGGGCACAGACCCTCTCAT 181 DNAJC5-1049 GGGTTCAACTAAATCCAGGAACGCCATCTCTGTGACTA  79 DNAJC19-475 GGATCTCCTTATATAGCAGCCAAGCCTTCCCTCCCAGTGA  93 AA DNAJC19-536 ATGCTCATCGGCGAATTATGAGCTGGAACGCATAAGAGAA 161 DNAJC24-628 CTCATTTTAATGGAAGATGGTATCACAAGAAATCAGT 176 DNAJC24-2172 CATCCAGATAAACAGAGTGGTCCCATATTTCTTAGAT 161 DNAJC24-1893 CAAAAGAAAGTATCTCATTCTAACTTCTTCTGCTTCATC 176 DNAJB12-1220 ACCGACTGTCAGAGACTATGCGGCCTCCAATTCCATTT 127

TABLE 2 Blastocyst and fertilization rates for genotypic classes ofDNAJC15 and DNAJC27 genes genotype (number of blastocyst DNAJC15 SNPovaries) rate ± SE P value SNP 85146(G > A) AA (202) 0.310 ± 0.025 0.014 AG (206) 0.281 ± 0.025  GG (53)  0.401 ± 0.040  SNP 85161(G > A)AA (202) 0.309 ± 0.023  0.022 AG (206) 0.283 ± 0.024  GG (61)  0.390 ±0.037  SNP 85216(A > G) GG (203) 0.312 ± 0.022  0.059 AG (201) 0.282 ±0.023  AA (63)  0.373 ± 0.036  SNP 85292(C > A) AA (204) 0.310 ± 0.023 0.012 AC (201) 0.279 ± 0.024  CC (62)  0.395 ± 0.037  SNP 85300(G > A)AA (208) 0.312 ± 0.023  0.066 AG (201) 0.280 ± 00.023 GG (63)  0.369 ±0.037  genotype (number of fertilization rate DNAJC27 SNP ovaries) ±SE Pvalue SNP 35728(G > A) AA (126) 0.621 ± 0.029 0.042 AG (257) 0.654 ±0.025 GG (103) 0.691 ± 0.030 SNP 36016(C > G) CC (129) 0.622 ± 0.0290.034 CG (251) 0.653 ± 0.026 GG (103) 0.693 ± 0.030 SNP 38867(A > G) AA(133) 0.626 ± 0.028 0.049 AG (246) 0.650 ± 0.026 GG (104) 0.693 ± 0.030

TABLE 3 Primers used for SNP identification, product sizes, and GenBankaccession numbers of amplified genes Amplicon GenBank PrimerPrimer sequence (5′-3′) (bp) Accession no. DNAJC15-1F: CCGGAGGTCTGCAAATGGG R: AACTGCTCGCCTGGTGCTGGTC 589 NC_007310 DNAJC15-2F: TCACTGAAAATCAGCCAATA R: CGTACAGAAGAGCCCCAT 569 NC_007310 DNAJC15-3F: AATTGCTTTATTACTTTAGCGG R: AGGGACCATGTCTGTTTTGT 647 NC_007310DNAJC15-4 F: AAAGTCCCTGTAGAGCTTAG R: ATAAAGGCACATCACAACTA 571 NC_007310DNAJC15-5 F: TCCTCCTGTCCTAGTTCTTG R: TTCATTATGCCCAAATCAGT 647 NC_007310DNAJC15-6 F: CCATCCACTTCAGAAAATTC R: GGGGAAAGATCAGTGCTAGAGT 620NC_007310 DNAJC19-1 F: T TTTCCGACCTAGTTTACGG R: ACTTCTACTTCACCACAGGGA670 NC_007299 DNAJC19-2 F: AGCCGCATACCTTTACAATG R: ATGGGTCACTTCAGATTCCT696 NC_007299 DNAJC19-3 F: TCCCAGAAGAACTGGGTTTG R: AGATGGAAGTCCCTGGCAGT496 NC_007299 DNAJC19-4 F: TTTAACTCACTGGAGGTAGG R: ACCAAAACAGCAAGTAGACC597 NC_007299 DNAJC19-5 F: AGGGATTGTTGATAACTGGA R: TTTATACCAACGCTTTGACT611 NC_007299 DNAJC24-1 F: GCTCGGCTGGAAACTTGA R: CGGTGGATGGCCCTCTAA 672NC_007313 DNAJC24-2 F: CCATTTCCTTCTCCACTAGT R: CTTTTGATTCCTGCTTTGAT 630NC_007313 DNAJC24-3 F: CCCTTCTGCTTTGTCCATC R: ACCACATTTCTGGGTTGCTC 538NC_007313 DNAJC24-4 F: GGAGACTTTTGGCCTAGTGT R: ATAAAGTTTTCAGGTGGGAA 637NC_007313 DNAJC24-5 F: TAAATAAATTCCCACCTGAA R: ACAATAGCCATGTTTTCTGA 649NC_007313 DNAJC24-6 F: GGCAACTGTAGAAAGGATAG R: TATAAAGAATAAGCACCACA 539NC_007313 DNAJC24-7 F: TGGTATTTATTATTGGTTTGGAC R: AGGAGAAAGGGATGACAAGG531 NC_007313 DNAJC27-1 F: CTCCTCCAGTTCCCTACCC R: A CAGCCCAGTAAGTTATCAGC665 NC_007309 DNAJC27-2F: TTGAAAACATACCCATATTTGG R: ATCACTAAAAGGAAGCTCTC 682 NC_007309DNAJC27-3 F: CTTCATCTTGCTCCTACTGTCC R: CAAAGGGTTGGTTCACTTCTG 647NC_007309 DNAJC27-4 F: AGGGACAGGGTAGAAGGC R: CAAACATGGCACCAGAAA 607NC_007309 DNAJC27-5 F: GGCAAACGTGATGAAGCC R: GACCTGGAGCCCAGCAAT 692NC_007309 DNAJC27-6 F: AGAGTAGGATCATAAGCCATTT R: TTCGGTGAAGGAGTAGTGTT532 NC_007309 DNAJC27-7 F: TGCAAGAGGTGTTCTGTTAT R: TTTCAGGGGTTCTACTATGT666 NC_007309 DNAJC27-8F: ACATAGTAGAACCCCTGAAAGT R: ATGATGCTGCAACAAGGAAA 500 NC_007309DNAJC27-9 F: TGCCAAGACAGGTGGGAAAT R: CAGGTAGGGTAAGGCGAATG 613 NC_007309DNAJC27-10 F: ATTCGCCTTACCCTACCTGA R: CTGGGAACTGAGCAAGACCTAA 644NC_007309 DNAJC27-11 F: CTTAGGTCTTGCTCAGTTCC R: CACAACATCTCCAAGTCCAG 668NC_007309 DNAJC27-12 F: ACTTGGAGATGTTGTGCTGC R: TATGAACCCCTCTTCCCTTT 565NC_007309 DNAJC27-13 F: GTGTCAGTATCTGTCCCCTAA R: TCTATGATCTCAGTCGGTAA681 NC_007309

1. An isolated nucleic acid molecule comprising a nucleotidecorresponding to a nucleotide at a first polymorphic position selectedfrom the group consisting of positions 85146, 85161, 85216, 85292, and85300 of the nucleic acid sequence shown in FIG. 1 (SEQ ID NO: 1), andat least 10 contiguous nucleotides of SEQ ID NO: 1 adjacent to the firstpolymorphic position, wherein position 85146 is guanine, position 85161is guanine, position 85216 is adenosine, position 85292 is cytosine, orposition 85300 is guanine; or an isolated nucleic acid moleculecomprising a nucleotide corresponding to a nucleotide at a secondpolymorphic position selected from the group consisting of positions35728, 36016, and 38867 of the nucleic acid sequence shown in FIG. 2(SEQ ID NO: 2), and at least 10 contiguous nucleotides of SEQ ID NO: 2adjacent to the second polymorphic position, wherein position 35728 isguanine, position 36016 is guanine, or position 38867 is guanine.
 2. Anucleic acid molecule according to claim 1, which comprises at least 10,at least 11, at least 12, at least 13, at least 14, or at least 15contiguous bases of SEQ ID NO: 1 adjacent to the first polymorphicposition, or of SEQ ID NO: 2 adjacent to second polymorphic position. 3.A nucleic acid molecule according to claim 1, which comprises at least20 contiguous bases of SEQ ID NO: 1 adjacent to the first polymorphicposition, or of SEQ ID NO: 2 adjacent to second polymorphic position. 4.An isolated nucleic acid molecule according to claim 1, which comprisesnot more than 150 contiguous nucleotides of SEQ ID NO: 1 adjacent to thefirst polymorphic position, or of SEQ ID NO: 2 adjacent to secondpolymorphic position.
 5. An isolated nucleic acid molecule according toclaim 1, which comprises not more than 100 contiguous nucleotides of SEQID NO: 1 adjacent to the first polymorphic position, or of SEQ ID NO: 2adjacent to second polymorphic position.
 6. An isolated nucleic acidmolecule according to claim 1, which comprises not more than 50contiguous nucleotides of SEQ ID NO: 1 adjacent to the first polymorphicposition, or of SEQ ID NO: 2 adjacent to second polymorphic position. 7.A nucleic acid molecule according to claim 1, wherein the first orsecond polymorphic site is within 4 nucleotides of the center of thenucleic acid molecule.
 8. A nucleic acid molecule according to claim 7,wherein the first or second polymorphic site is at the center of thenucleic acid molecule.
 9. A nucleic acid molecule according to claim 1,wherein the first or second polymorphic site is at the 3′-end of thenucleic acid molecule.
 10. An array of nucleic acid molecules comprisingat least two nucleic acid molecules according to claim
 1. 11. A kitcomprising a nucleic acid molecule of claim 1, and a suitable container.12. A method for detecting single nucleotide polymorphism in a bovineheat-shock protein (HSP) gene, wherein the HSP gene comprises a nucleicacid sequence homologous to SEQ ID NO: 1 or SEQ ID NO: 2, the methodcomprising obtaining a sample of the HSP gene from a bovine animal,determining the identity of a nucleotide of the HSP gene at a positioncorresponding to position 85146, position 85161, position 85216,position 85292, or position 85300 of SEQ ID NO: 1; or to position 35728,position 36016, or position 38867 of SEQ ID NO: 2, and comparing thenucleotide identity of the position respectively to the nucleotideidentity at a corresponding position of SEQ ID NO: 1 or SEQ ID NO: 2.13. A method for genotyping a bovine cell, comprising obtaining anucleic acid sample from said cell and determining the identity of atleast one nucleotide corresponding to a position selected from the groupconsisting of positions 85146, 85161, 85216, 85292, and 85300 of SEQ IDNO: 1, and positions 35728, 36016, and 38867 of SEQ ID NO:
 2. 14. Amethod according to claim 13, wherein the bovine cell is an adult cell,an embryo cell, a sperm, an egg, a fertilized egg, or a zygote.
 15. Amethod according to claim 13, wherein the identity of the nucleotide isdetermined by sequencing the HSP gene, or a relevant fragment thereof,isolated from the cell.
 16. A method according to claim 15, wherein thegene or a relevant fragment thereof is isolated from the cell viaamplification by the polymerase chain reaction (PCR) of genomic DNA ofthe cell, or by RT-PCR of the mRNA of the cell.
 17. A method accordingto claim 15, wherein both copies of the gene in the cell are genotyped.18. A method for progeny testing of cattle, the method comprisingcollecting a nucleic acid sample from said progeny, and genotyping saidnucleic sample according to claim
 13. 19. A method for selectivelybreeding cattle using a multiple ovulation and embryo transfer procedure(MOET), the method comprising superovulating a female animal, collectingeggs from said superovulated female, in vitro fertilizing said eggs froma suitable male animal, implanting said fertilized eggs into otherfemales allowing for an embryo to develop, and genotyping saiddeveloping embryo according to claim 13, and terminating pregnancy ifthe developing embryo does not have at least one polymorph of a HSP geneselected from the group consisting of a first polymorph selected fromthe group consisting of guanine at position 85146, guanine at position85161, adenosine at position 85216, cytosine at position 85292, guanineat position 85300 of SEQ ID NO: 1; and a second polymorph selected fromthe group consisting of guanine at position 35728, guanine at position36016, and guanine at position 38867 of SEQ ID NO:
 2. 20. The methodaccording to claim 19, wherein pregnancy is terminated if the developingembryo does not have either the first polymorph or the second polymorph.21. The method according to claim 19, wherein pregnancy is terminated ifthe developing embryo is not homozygous in both the first polymorph andthe second polymorph.
 23. A method for selecting a cattle as a breeder,wherein the cattle is genotyped according to claim 13, and is onlyselected for breeding purpose if it comprises at least one polymorphselected from the group consisting of the first polymorph which isselected from guanine at position 85146, guanine at position 85161,adenosine at position 85216, cytosine at position 85292, guanine atposition 85300 of SEQ ID NO: 1; and a second polymorph selected from thegroup consisting of guanine at position 35728, guanine at position36016, and guanine at position 38867 of SEQ ID NO:
 2. 24. The methodaccording to claim 23, wherein the cattle animal is selected only if itcomprises both the first and second polymorphs.
 25. The method accordingto claim 24, wherein the cattle animal is selected only if it ishomozygous with regard to both the first and second polymorphs.